Contents

Overview & Purpose

Text describing the image

By modifying the input and output for the Lac switch, it may be possible to produce materials such as plastics. The switch could be triggered by another environmental factor other than [IPTG], and instead of producing GFP, more useful materials like plastic could be an output. Currently, production of plastics is a very energy intensive process, and by using bacteria for the production, we can save energy and limit waste into the environment.

The IPTG-input/fluorescent protein-output is proof of concept for the production of synthetic materials such as plastic being created by bacteria. In the original project, a synthetic compound was utilized to trigger the metabolic pathway for the degradation of lactose. Using synthetic biology, we can splice together different genetic features to create an entirely new metabolic response. We can find a promoter that responds to a different input. In the natural Lac-operon, the cell produces proteins for the breakdown of lactose. We modified the natural process by initiating the production of GFP instead of the functional proteins found in the original process. It seems that if we are able to control our protein output, we can produce synthetic products as well.

Background

Diagram of Lac operon and how it functions without lactose present (top) and with lactose present (bottom)

The natural Lac-operon has 2 controls that tightly regulate the production of the proteins necessary for the breakdown of lactose. In the presence of glucose, the Lac Operon inhibits the production of those proteins. When glucose is present, the lac repressor is bound to the operator, prohibiting the transcription of the proteins. In the presence of lactose, lactose is able to bind to the lac repressor initiating a conformation change in the repressor protein, causing it to release and allow for transcription. This, however, is not the only control in place. It is incredibly energy intensive to produce the proteins necessary for the breakdown of lactose, so when there is glucose present, the glucose will be metabolized first, even with some lactose present. This is accomplished by the presence of a second regulatory device, cyclic AMP (cAMP). cAMP is present only when there are very low levels of glucose found in the environment. cAMP serves as an activator and binds to RNA polymerase allowing for transcription of the output proteins.

In the synthetic [IPTG] induced Lac-operon, IPTG serves the same function as lactose, it binds to the repressor causing a conformational change, allowing for transcription of proteins. In the natural Lac-Operon, the proteins were functional, and necessary for the breakdown of lactose. In our synthetic system, green fluorescent protein is produced instead. This protein serves as an indicator, allowing for visual verification that the lac switch is operational.

Design: Our genetic circuit

OUR GENE SWITCH:

AND gate Gene Switch: Both [IPTG] AND [Low Glucose Levels] conditions must be met for GFP production

Truth table describing inputs required to produce an output

The functionality of our genetic switch resembles that of an "AND" logic gate: the device requires two conditions to be true in order for an output to be produced. One conditional requirement is that IPTG must be present in the device's environment. When IPTG is present, it binds to the LacI repressor, thus allowing for transcription to continue. The other conditional requirement is that glucose levels in the device's environment must be low. Glucose levels inversely affect production of cyclic AMP (cAMP): when glucose levels are low, cAMP production increases and when glucose levels are high, cAMP production decreases. cAMP binds to catabolite activator protein (CAP) to form the CAP-cAMP complex. In order for this complex to form, cAMP must be present and, thus, glucose levels must be low.[1] The CAP-cAMP complex is an input that our device requires in order to produce an output.

In the natural lac operon, the CAP-cAMP complex leads to enhanced activation of gene expression from the lac operon. However, if glucose is present, cAMP levels will in turn be low and the host will preferentially metabolize glucose even if lactose is present.

Building: Assembly Scheme

Diagram of Lac operon and how it functions without lactose present (top) and with lactose present (bottom)

Testing: Modeling and GFP Imaging

We used a previously published synthetic switch, developed by Ceroni et al., to understand how our system could potentially be modeled and simulated. Mathematical modeling is a method of representing via the language of math how a system is expected to behave. The various components of a system, referred to as system parameters, are assigned variable names. These variables hold values that can be adjusted during testing without incurring what would otherwise be additional production costs. Modeling is very beneficial in that multiple tests and simulations can be performed on the system so that a predictable pattern of behavior can be developed, thus reducing production costs.

We modeled our device after the Ceroni switch, using similar variables and parameters to represent the various components and processes of our device. The graphic to the right depicts these components and the variables used to represent them in a mathematical model. The following table contains the variables and parameters used both in our model and the Ceroni et al., model.

Variables and parameters used by both our device's model and the Ceroni model

Variable

Description

Units

I

IPTG Concentration

mM

G

Concentration of GFP protein

molecules/cell

LF

Free LacI molecules

molecules/cell

LI

LacI molecules bound to IPTG

molecules/cell

MG

mRNA molecules of GFP

molecules/cell

ML

mRNA molecules of LacI

molecules/cell

λG/L

Protein degradation rate

minutes-1

λMG/L

mRNA degradation rate

minutes-1

αG

GFP rate of synthesis

minutes-1

AL

LacI rate of synthesis

minutes-1

αMG

GFP transcription rate

minutes-1

αML

LacI transcription rate

minutes-1

AN INTERACTIVE MODEL
We used a model of the natural Lac operon to understand how changing the parameter values changes the behavior of the system.

COLLECTING IMPERICAL VALUES TO IMPROVE THE MODEL
We explored how one technique, imaging via microscopy could be used to determine the production rate of an output protein, in this case GFP in yeast, could be used to determine a "real" value for maximum GFP production rate under our own laboratory conditions.

Ideally, the GFP production rate measured by this method could be entered as a value for [which parameter] in the Ceroni et al. model.

Human Practices

Danger of Chemicals in Farmlands

//////////////NOTES (RK)//////////////
- switch that could detect tumor antigens (input)
- If we were to make the switch dependent on a tumor antigen input, the system could potentially detect a cancer cell and switch on, at which point it could produce synthetic antibodies as outputs, which could potentially speed up the host's immunologic response. Additionally, researches could experiment using different output proteins to see which ones would be most effective in neutralizing cancer cells while reducing damage to neighboring cells.

- By switching the promoters at the beginning of the system to take as an input
-
-explain who is in each stakeholder quadrant and why, and how you engage with people or within each quadrant
-or gap analysis

Our Team

Your Name

My name is Jennifer Sherwood, and I am a senior majoring in chemical engineering. I am taking BME 494 because I am interested in learning new ways to engineer biology. An interesting fact about me is that I enjoy playing golf and creating stained glass lamps in my spare time.

Randle Kuehner

My name is Randle Kuehner, and I am a part-time senior majoring in biomedical engineering. I am taking BME 494 because synthetic biology sounded incredibly interesting and I didn't really know anything about it prior to this semester. An interesting fact about me is that, from Phoenix, I drove to the Arctic Ocean at Deadhorse, AK and back in July 2010.

Your Name

My name is ###, and I am a ### majoring in ###. I am taking BME 494 because ###. An interesting fact about me is that ###.